Genomic study of Acinetobacter baumannii strains co-harboring blaOXA-58 and blaNDM-1 reveals a large multidrug-resistant plasmid encoding these carbapenemases in Brazil

Introduction Acinetobacter baumannii contributes significantly to the global issue of multidrug-resistant (MDR) nosocomial infections. Often, these strains demonstrate resistance to carbapenems (MDR-CRAB), the first-line treatment for infections instigated by MDR A. baumannii. Our study focused on the antimicrobial susceptibility and genomic sequences related to plasmids from 12 clinical isolates of A. baumannii that carry both the blaOXA-58 and blaNDM-1 carbapenemase genes. Methods Whole-genome sequencing with long-read technology was employed for the characterization of an A. baumannii plasmid that harbors the blaOXA-58 and blaNDM-1 genes. The location of the blaOXA-58 and blaNDM-1 genes was confirmed through Southern blot hybridization assays. Antimicrobial susceptibility tests were conducted, and molecular characterization was performed using PCR and PFGE. Results Multilocus Sequence Typing analysis revealed considerable genetic diversity among blaOXA-58 and blaNDM-1 positive strains in Brazil. It was confirmed that these genes were located on a plasmid larger than 300 kb in isolates from the same hospital, which also carry other antimicrobial resistance genes. Different genetic contexts were observed for the co-occurrence of these carbapenemase-encoding genes in Brazilian strains. Discussion The propagation of blaOXA-58 and blaNDM-1 genes on the same plasmid, which also carries other resistance determinants, could potentially lead to the emergence of bacterial strains resistant to multiple classes of antimicrobials. Therefore, the characterization of these strains is of paramount importance for monitoring resistance evolution, curbing their rapid global dissemination, averting outbreaks, and optimizing therapy.


Introduction
Acinetobacter baumannii has emerged as one of the most significant and challenging pathogens of this century, primarily due to its involvement in multidrug-resistant (MDR) hospital-acquired infections (Zhang et al., 2021).The most common clinical presentations linked to this pathogen include ventilator-associated pneumonia (VAP) and bloodstream infections (Palmieri et al., 2020).The management of Acinetobacter infections is notably difficult due to its exceptional capacity to develop resistance to nearly all classes of antimicrobials currently used in clinical settings, particularly carbapenems (Ghaly et al., 2020), which are the treatment of choice for MDR A. baumannii (MDR-Ab) infections.However, the distressing increase in carbapenem-resistant A. baumannii (CRAB) strains globally has significantly limited treatment alternatives and considerably increased morbidity and mortality rates (Kyriakidis et al., 2021).Importantly, CRAB strains often demonstrate co-resistance to other antibiotic classes, further complicating therapeutic approaches (Nodari et al., 2020).
Carbapenem resistance in Acinetobacter spp.can also occur.However, it is less common due to the production of metallo-βlactamases (MBL), such as imipenemase (IMP), Verona integronencoded metallo-β-lactamase (VIM), and New Delhi metallo-β-lactamase (NDM) (Anggraini et al., 2022).Notably, NDM-1 is prevalent in Klebsiella pneumoniae and Escherichia coli species, whereas Acinetobacter spp. is known as an intermediate reservoir.However, NDM-1-producing Acinetobacter strains have been reported globally due to the high horizontal transferability of the plasmids carrying the bla NDM gene and several additional resistance mechanisms, restricting treatment options (Vasudevan et al., 2022).
The co-occurrence of two distinct carbapenemase-encoding genes is a particularly alarming mechanism of antimicrobial resistance, as it typically results in higher resistance to β-lactams and is often associated with increased mortality rates (Oinuma et al., 2016;Vasudevan et al., 2022).
In this study, we examined antimicrobial susceptibility and investigated the genomic sequences associated with plasmids from A. baumannii clinical isolates carrying the bla OXA-58 and bla NDM-1 genes, with the aim of gaining a deeper understanding of the molecular basis and evolutionary dynamics of the antimicrobial resistance of isolates circulating in Brazil.

Antimicrobial susceptibility testing
The antimicrobial susceptibility profile was performed and interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines (European Committee on Antimicrobial Susceptibility Testing (EUCAST), 2023).Antimicrobial susceptibility testing (AST) of A. baumannii isolates for amikacin, gentamicin, ciprofloxacin, trimethoprim-sulfamethoxazole, tobramycin, levofloxacin, and meropenem was performed using the Kirby-Bauer disk-diffusion method, as described previously (Bauer et al., 1966).The broth dilution method was used to determine the susceptibility to colistin.

Pulsed-field gel electrophoresis
All the isolates were analyzed using PFGE, as described previously, with minor modifications (Bou et al., 2000).Bacterial cells were embedded in agarose plugs and digested by ApaI (Invitrogen) at 37°C for 3 h.Electrophoresis was performed on 1.1% agarose gel (SeaKem ® Gold Agarose, Lonza) in 0.4× Tris-borate-EDTA buffer using a CHEF-DR III System (Bio-Rad) apparatus.Images of banding patterns obtained were processed using BioNumerics software (version 6.6; Applied Maths).Similarities were calculated using both Dice coefficients, and the unweighted pair-group method using arithmetic averages (UPGMA) was applied for cluster analysis.The tolerance and optimization were set at 1.5% each.Strains were considered epidemiologically related if they had ≥90% genetic similarity.
Plasmids were analyzed using S1 nuclease (Invitrogen) digestion followed by Southern blot hybridization for all 12 strains.After transfer to Amersham Hybond-N+ membranes (GE Healthcare), the genomic DNA was hybridized with the bla OXA-58 and bla NDM-1 probes as described in the DIG-DNA labeling and detection kit (Roche Diagnostics, Germany).

Whole-genome sequencing
Only CCBH31258 was subjected to long-read sequencing by Oxford Nanopore Technologies ® (ONT).Genomic DNA was extracted using the DNeasy ® PowerSoil ® Pro Kit (Qiagen), following the manufacturer's recommendations.The barcoding sequencing library was prepared according to the protocol for native barcoding genomic DNA using the Ligation Sequencing SQK-LSK109 and Native Barcoding Expansion 1-12 EXP-NBD104 kits (Oxford Nanopore Technologies ® ), as well as the NEB Blunt/TA Ligase Master Mix, NEBNext ® Quick Ligation Reaction Buffer, and NEBNext ® Companion Module for Oxford Nanopore Technologies ® Ligation Sequencing.
Based on the PFGE analysis, all isolates except CCCBH31258 were subjected to Illumina whole-genome sequencing.Genomic DNA from each strain from the overnight culture was extracted using the QIAamp DNA mini kit (Qiagen), following the manufacturer's instructions.A tagmentation library from genomic DNA was made using the Nextera XT DNA Sample Preparation Kit (Illumina, San Diego, CA, United States), and the 250-bp paired-end reads were sequenced on the Miseq system (Illumina, San Diego, CA, United States).

Assembly
The de novo assembly for CCBH31258 long reads was generated using the Flye assembler (Kolmogorov et al., 2019).For the short reads from the other seven strains, Trimmomatic was used at the trimming step (ILLUMINACLIP:NexteraPE-PE.fa:2:30:10AVGQUAL:20 MINLEN:50) (Bolger et al., 2014).The genome de novo assembly was performed using Unicycler (v0.4.9), conservative mode (Wick et al., 2017).Short reads were mapped against plasmids from CCBH31258 (pCCBH31258) using minimap2 and SAMtools (Li et al., 2009;Li, 2018).For strains that read coverage for all pCCBH31258, a plasmid consensus sequence was generated using Pilon, and VCF output was checked (Walker et al., 2014).Furthermore, contigs were mapped against the plasmid consensus using minimap2, and the mapped contigs were excluded to obtain only chromosome contigs.Contigs from the other strains were extended by AlignGraph (Bao et al., 2014), using pCCBH31258 as a reference.For each strain, mapped reads (SAM files) using pCCBH31258 as a reference were compared.

Annotation
Draft genomes were submitted to GenBank and annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP).The web application CABGen was used to access coverage estimation; species confirmation; MLST mapping; searches for genes related to AMR, virulence, and plasmids; and detection of point mutations in specific AMR genes (Duré et al., 2022).Resistance genes were confirmed using raw reads as input for ResFinder software on the Center for Genomic Epidemiology website.Insertion sequence annotation was improved in the figures using ISFinder (Siguier, 2006).MOB-suite was applied to the assembled plasmid to predict the Inc. group, relaxase typing, and conjugation potential (Robertson and Nash, 2018).PdifFinder analyses were adopted for the annotation of pdif sites and pdif-ARGs modules in the plasmid (Shao et al., 2023).

Antimicrobial susceptibility profile
The isolates exhibited a resistance profile to the majority of the antimicrobials tested, with the highest resistance rates to meropenem (100%), gentamicin (100%), trimethoprim-sulfamethoxazole (83%), and tobramycin (83%).Colistin was the most active agent tested, with only two resistant isolates, one from Bahia and the other from Amazonas (Figure 1).

Pulsed-field gel electrophoresis
The 12 strains were grouped into six PFGE clusters (A, B, C, D, E, and F), and the most frequent pulsetype was pattern F (six strains from the same hospital in Bahia), followed by pattern E (2 strains from two different hospitals in Amazonas).In Bahia and Amazonas, two other pulsetypes were found (A and B in Bahia and C and D in Amazonas).Southern blot hybridization showed that bla OXA-58 and bla NDM-1 genes were located on a plasmid with approximately 320 kb for clone B and clone F strains, which were isolated in the Bahia state, in the same hospital.Furthermore, two isolates from the Amazonas state, belonging to clone E, showed labeled bands with similar weights (CCBH31624 and CCBH32292).Two strains from Amazonas (CCBH33291 and CCBH33294), isolated in the same hospital, showed the same labeled band of approximately 178 kb, although they belong to different clones (C and D) (Figure 1; Supplementary material).

Whole-genome sequencing
The de novo assembly for CCBH31258 (pulsotype F) long reads generated two contigs: one chromosome with 3,706,655 bp and one circularized plasmid with 340,418 bp (GenBank accession
The bla OXA-58 was inserted between a complete ISAba3 upstream and a truncated ISAba3 downstream, followed by a complete ISApi2.This structure belongs to a pdif-ARGs module in pCCBH31258, surrounded by inversely oriented pdif sites (XerC/D and XerD/C).Surrounding bla OXA-58 , araC family gene and genes related to efflux systems (adeR, adeS, MFS transporter, and multidrug efflux RND) were identified.Macrolide resistance genes, msr(E) and mph(E), were also located next to bla OXA-58 (Figure 2A).
Although the weight estimation derived from hybridization results suggested that CCBH31624, isolated in Amazonas, harbored a plasmid similar to pCCBH31258, sequencing results indicated distinct gene sets (Figure 1).According to the mapping results and gene content, CCBH31624 and CCBH32292 (both pulsotype E, ST464) appear to carry a plasmid with a genomic background different from pCCBH31258, despite the similar weight observed in the Southern blot.For CCBH29641 (pulsotype A, ST739), retrieved from a different hospital in Bahia, the mapping results also demonstrated a genomic background distinct from pCCBH31258.Two sequenced strains from the same hospital in Amazonas (CCBH33291 and CCBH33294), belonging to ST10 and ST575, respectively, exhibited minimal similarities with pCCBH31258.For all these five strains, Tn125 harboring bla NDM-1 was incomplete (Figure 2A).
Consequently, for STs other than ST374, it could not be confirmed by sequencing that bla OXA-58 and bla NDM-1 were inserted in the same plasmid, despite indications from Southern blot results.Given this, the contigs output from Align-Graph was adopted.For all isolates in this study, only the upstream insertion ISAba125 and downstream bleomycin gene were conserved surrounding bla NDM-1 , and only the ISAba3 upstream and downstream regions were conserved for bla OXA-58 (Figure 2A).

Discussion
Acinetobacter baumannii exhibits an extraordinary ability to develop resistance to multiple antimicrobial agents, particularly through mobile genetic elements, such as plasmids, that harbor antimicrobial resistance determinants.The prevalence of multidrugresistant A. baumannii (MDR-Ab) infections in hospital settings is a significant health concern, especially regarding last-resort antibiotics such as carbapenems.In the present study, all bla OXA-58 and bla NDM-1 positive strains were resistant to meropenem and exhibited the MDR phenotype, aligning with the global increase in MDR-Ab infections (Nguyen and Joshi, 2021).
Carbapenem-resistant A. baumannii (CRAB) is a therapeutic challenge because few currently available antibiotics are active against CRAB (Bartal et al., 2022).Carbapenem resistance makes colistin the last-resort antimicrobial to treat MDR-Ab infections.However, overuse of this antimicrobial to treat CRAB infections may have contributed to the increased prevalence of colistin-resistant A. baumannii (Kurihara et al., 2022).Although most of the strains in the current study were susceptible to colistin, resistance was observed in two isolates.
CRAB is generally associated with a wide range of co-resistance to other classes of antimicrobial agents.The production of OXA-type carbapenemases is the main mechanism of carbapenem resistance in A. baumannii.These carbapenemases are encoded by the bla OXA genes, which are usually carried by highly transmissible plasmids (Nguyen and Joshi, 2021).In Brazil, bla OXA-23 is the most widely disseminated OXA-type carbapenemase in CRAB, followed by bla OXA-143 , while bla OXA- 58 is less common (de Souza Gusatti et al., 2012;de Oliveira et al., 2019;Matos et al., 2019).
OXA-58 was first described in 2003 in France; since then, it has been widely reported worldwide, being associated with outbreaks of nosocomial infections (Moro et al., 2008;Ozen et al., 2009;de Souza Gusatti et al., 2012).OXA-58 hydrolyzes carbapenems at low levels; however, expression can be increased by the presence of insertion sequences, such as ISAba3, resulting in resistance to carbapenems (Walther-Rasmussen and Høiby, 2006;Evans and Amyes, 2014).In the present study, all representative isolates sequenced showed complete ISAba3 upstream of the bla OXA-58 gene, suggesting its higher expression.The bla OXA-58 gene was inserted in a pdif module flanked by inversely oriented pdif sites in pCCBH31258, suggesting that this gene was probably mobilized and mediated by pdif sites using a XerC-XerD recombination system (Li et al., 2022a).
The emergence of NDM in CRAB has also become an important public health issue, which remains a major challenge for the treatment of infectious diseases.NDM is reported in most regions of the world due to its rapid dissemination since the gene encoding NDM is often carried by transferable plasmids (Villacís et al., 2019).pCCBH31258 analyzed in this study revealed that the bla NDM-1 gene was located between two copies of the ISAba125 element, and all strains carried the ble MBL (bleomycin resistance) gene, which has always been identified downstream of the bla NDM-1 gene.Several studies reported that the genetic environment of the bla NDM-1 gene presents a conserved structure, where this gene is located between two copies of the ISAba125 element, forming a transposon named Tn125 (Dortet et al., 2014;Pagano et al., 2016).However, it could be observed in this study that the Tn125 complete structure was conserved only for the strains that harbored the complete plasmid characterized here.
Our research describes 12 strains of carbapenem-resistant Acinetobacter baumannii (CRAB) concurrently carrying bla OXA-58 and bla NDM-1 , representing 1% of all Acinetobacter species received by the laboratory during the period.The coexistence of these carbapenemase genes in CRAB has been infrequently reported.There have been four instances of CRAB isolates co-producing OXA-58 and NDM-1 from Algeria (Ramoul et al., 2016), Japan (Oinuma et al., 2016), Malaysia (Alattraqchi et al., 2021), and China (Liu et al., 2021).Only the strains isolated in Malaysia and China have the bla OXA-58 and bla NDM-1 genes located on the same plasmid (Alattraqchi et al., 2021;Liu et al., 2021) (Figure 2B), while in the Algerian isolate, the genes were chromosomally located (Ramoul et al., 2016), and the genome of the Japanese strain is a draft (Oinuma et al., 2016).Other non-baumannii Acinetobacter carrying bla OXA-58 and bla NDM-1 genes have been reported from Malaysia (Alattraqchi et al., 2021), China (Zhou et al., 2015;Chen et al., 2019;Li et al., 2022a,b), and Palestine (Regeen et al., 2014).Of these reports, three isolates were sequenced and found to carry these genes on the same plasmids (Alattraqchi et al., 2021;Li et al., 2022a,b) (Figure 2B).To the best of our knowledge, this is the first study reporting A. baumannii strains in Brazil co-harboring bla OXA-58 and bla NDM-1 genes.Genomic analysis revealed that pCCBH31258 harbored genes coding for proteins with high-level similarity to components of the Dot/Icm type IV secretion system from species of Legionella and Coxiella (type IVB) (Segal et al., 2005).
It could be noted that there is a high level of ST diversity associated with the co-occurrence of bla OXA-58 and bla NDM-1 in Brazil, which suggests that this co-occurrence does not relate exclusively to the clone.MLST analysis showed that five different sporadic STs were present in 12 wholegenome sequenced strains: ST10, ST374, ST464, ST739, and ST575.Interestingly, none of the STs described here belonged to the main clonal complexes CC1, CC15, CC25, and CC79 predominant in South America or to CC2, which is the most prevalent clonal lineage in the world (Rodríguez et al., 2018).The same diversity of plasmids co-harboring bla OXA-58 and bla NDM-1 can also be highlighted, considering isolates from other countries (Alattraqchi et al., 2021;Liu et al., 2021;Li et al., 2022a,b).
The most frequent ST was ST374, including six isolates from the same hospital, and all these strains carried the same plasmid cohabiting bla OXA-58 /bla NDM-1 .The A. baumannii ST374 has rarely been reported (Fedrigo et al., 2022), and only one study from Tanzania reported a meropenem-resistant isolate, belonging to ST374, which was found to harbor bla NDM-1 in a chromosomally located composite transposon Tn125 (Moyo et al., 2021), while our observations revealed a plasmid localization for the bla NDM-1 gene.
A very similar plasmid to the one characterized here was observed in an isolate in the same hospital, belonging to ST464 (CCBH31950).Hybridization approaches suggest that another plasmid (~178 kb) is responsible for the mobilization of the two carbapenemase-encoding genes in strains from different clones but isolated in the same hospital in Amazonas state.Horizontal transfer enables different clonal lineages to acquire the same accessory genetic elements (Valenzuela et al., 2007).Furthermore, the mobilization of both genes on the same plasmid may develop an increasing number of OXA-58/NDM-1 co-producer isolates.This scenario is favored by the high plasticity that the genome of A. baumannii has in acquiring, retaining, and disseminating antimicrobial resistance genes, especially in plasmids, which play a key role in harboring and transferring antibiotic-resistant genes (Roca et al., 2012).

Conclusion
Our study delineates a large plasmid harboring OXA-58 and NDM-1 carbapenemases, which have disseminated among clonal strains isolated during 2021 in the same hospital.These strains have accumulated other antimicrobial resistance genes.Furthermore, other genetic contexts have also demonstrated the capacity to disseminate bla OXA-58 and bla NDM-1 .Our limited access to ONT sequencing prevented the complete genomic characterization of all plasmids harboring bla OXA-58 and bla NDM-1.However, this study reports the first identification of A. baumannii strains co-harboring these carbapenemase genes in Brazil.The co-occurrence of the carbapenemase genes bla OXA-58 and bla NDM-1 in clinical strains of A. baumannii is relatively uncommon.To the best of our knowledge, the 12 co-producing strains identified in this study represent the highest number reported in a single investigation.The spread of plasmids harboring critical antimicrobial resistance genes may facilitate the emergence of bacterial strains resistant to several classes of antibiotics, thereby presenting new challenges for the treatment of infectious diseases.Thus, the results presented highlight the propensity of A. baumannii to acquire and accumulate resistance genes to significant antimicrobial classes utilized in clinical practice.The characterization of MDR-CRAB strains is crucial for monitoring the evolution of resistance, with the aim of containing their rapid global spread, preventing outbreaks, and optimizing therapy.